Micelle-based brain-targeted drug delivery enabled by a nicotine acetylcholine receptor ligand.

The blood–brain barrier (BBB) is the key challenge in the development of drugs for diseases of the central nervous system (CNS). The BBB prevents drugs or drug delivery systems from reaching the site of disease because of tight junctions and lack of fenestration. To circumvent this problem, the receptors that are highly expressed on the capillary endothelium of the brain, such as nicotine acetylcholine receptors (nAChRs), have been exploited to facilitate BBB crossing and intracranial transport of drug delivery systems. nAChRs are ligand-gated ion channels that are expressed mainly at the neuromuscular junction of the CNS, including the brain capillary endothelial cells. The extensive expression of nAChRs in the brain and susceptibility to the inhibition by peptide neurotoxins and neurotropic viral proteins endow them with the ability to mediate peptidebased transvascular delivery of various therapeutic agents to the brain. Herein, we report the design of a 16-residue peptide, derived from the loop II region of the snake neurotoxin candoxin, that binds to nAChRs with high affinity. This peptide, termed CDX, enabled drug delivery to the brain when conjugated to paclitaxel-loaded micelles. As a result, tumor growth in intracranial glioblastoma bearing mice was inhibited and their survival was prolonged. Snake neurotoxins are members of the “three-finger toxin” superfamily characterized by three adjacent loops arranged in a flat, leaflike structure. These toxins are known to bind through the second loop to nAChRs with high affinity and selectivity. Candoxin from the Malayan krait Bungarus candidus consists of a single polypeptide chain of 66 amino acid residues with five disulfide bridges, and antagonizes a7 neuronal nAChRs in nanomolar concentrations with poor reversibility. As was shown previously by western blot analysis, and confirmed by using immunocytochemical staining (Figure S8 in the Supporting Information), the a7 neuronal nAChR is richly expressed in primary brain capillary endothelial cells, and is thus ideally suited for candoxin-mediated, brain-targeted drug delivery. For this study, we designed and evaluated three short peptides derived from the loop II region of candoxin, FKESWREARGTRIERG (CDX), SWREARGTRI (Pocket_CDX), and disulfide bridged CFKESWREARGTRIERGC (Cyclo_CDX). To investigate whether or not the candoxin-derived peptides are capable of interacting with rat neuronal nAChRs, we performed a competitive binding assay where different concentrations of peptide competed for receptor binding with radiolabeled I-a-bungarotoxin, which is a potent antagonist of a7 neuronal nAChRs. All three peptides functioned as competitive antagonists of neuronal nAChRs in a dose-dependent manner (Figure S2). CDX displayed a Ki value of 0.187 mm, which is approximately 20– 40 times lower than those of Pocket_CDX and Cyclo_CDX (Table 1). Not surprisingly, CDX is substantially less potent than candoxin in nAChRs binding. The difference in potency is likely attributable, at least in part, to a loss of entropy for CDX, as it is unstructured in aqueous solution, as indicated by circular dichroism spectroscopic analysis (Figure S3).